Thirteen novel canine dog leukocyte antigen-88 alleles identified by sequence-based typing.

Major histocompatibility complex (MHC) genes in mammals include highly polymorphic class I and class II genes that are critical for donor-recipient matching for transplantation. Dogs have served as an effective, directly translatable model for stem/progenitor cell transplantation. Previous analyses of MHC class I genes in dogs point to a single highly polymorphic gene, dog leukocyte antigen (DLA)-88, as an important factor in the success or failure of hematopoietic stem cell transplants. Fifty-nine DLA-88 alleles have been identified and reported so far. Here, we extend this list by presenting 13 novel DLA-88 alleles found in domestic dogs.

Canine DLA-79 gene: an improved typing method, identification of new alleles and its role in graft rejection and graft-versus-host disease.

Developing a preclinical canine model that predicts outcomes for hematopoietic cell transplantation in humans requires a model that mimics the degree of matching between human donor and recipient major histocompatibility complex (MHC) genes. The polymorphic class I and class II genes in mammals are typically located in a single chromosome as part of the MHC complex. However, a divergent class I gene in dogs, designated dog leukocyte antigen-79 (DLA-79), is located on chromosome 18 while other MHC genes are on chromosome 12. This gene is not taken into account while DLA matching for transplantation. Though divergent, this gene shares significant similarity in sequence and exon-intron architecture with other class I genes, and is transcribed. Little is known about the polymorphisms of DLA-79 and their potential role in transplantation. This study was aimed at exploring the reason for high rate of rejection seen in DLA-matched dogs given reduced intensity conditioning, in particular, the possibility that DLA-79 allele mismatches may be the cause. We found that about 82% of 407 dogs typed were homozygous for a single, reference allele. Owing to the high prevalence of a single allele, 87 of the 108 dogs (∼80%) transplanted were matched for DLA-79 with their donor. In conclusion, we have developed an efficient method to type alleles of a divergent MHC gene in dogs and identified two new alleles. We did not find any statistical correlation between DLA-79 allele disparity and graft rejection or graft-versus-host disease, among our transplant dogs.

Canine bone marrow-derived mesenchymal stromal cells suppress alloreactive lymphocyte proliferation in vitro but fail to enhance engraftment in canine bone marrow transplantation.

Stable mixed hematopoietic chimerism has been consistently established in dogs who were mildly immunosuppressed by 200 cGy of total body irradiation (TBI) before undergoing dog leukocyte antigen (DLA)-identical bone marrow (BM) transplantation and who received a brief course of immunosuppression with mycophenolate mofetil (28 days) and cyclosporine (35 days) after transplantation. However, when TBI was reduced from 200 to 100 cGy, grafts were nearly uniformly rejected within 3-12 weeks. Here, we asked whether stable engraftment could be accomplished after a suboptimal dose of 100 cGy TBI with host immunosuppression enhanced by donor-derived mesenchymal stromal cells (MSCs) given after transplantation. MSCs were cultured from BM cells and evaluated in vitro for antigen expression. They showed profound immunosuppressive properties in mixed lymphocyte reactions (MLRs) in a cell dose-dependent manner not restricted by DLA. MSC and lymphocyte contact was not required, indicating that immunosuppression was mediated by soluble factors. Prostaglandin E2 was increased in culture supernatant when MSCs were cocultured in MLRs. The addition of indomethacin restored lymphocyte proliferation in cultures containing MSCs. MSCs expressed CD10, CD13, CD29, CD44, CD73/SH-3, CD90/Thy-1, and CD106/VCAM-1. For in vivo studies, MSCs were injected on the day of BM grafting and on day 35, the day of discontinuation of posttransplantation cyclosporine. MSCs derived from the respective BM donors failed to avert BM graft rejection in 4 dogs who received DLA-identical grafts after nonmyeloablative conditioning with 100 cGy TBI in a time course not significantly different from that of control dogs not given MSCs. Although the MSCs displayed in vitro characteristics similar to those reported for MSCs from other species, their immunosuppressive qualities failed to sustain stable BM engraftment in vivo in this canine model.

An improved method for dog leukocyte antigen 88 typing and two new major histocompatibility complex class I alleles, DLA-88*01101 and DLA-88*01201.

Development of preclinical dog models of solid organ and hematopoietic transplantation is critically dependent upon characterization of the polymorphic major histocompatibility complex class I and class II loci. While the class II alleles are easily typed as the polymorphic positions reside on a single exon, typing the class I locus is tedious. We have improved the class I typing method by designing improved primers and adopting alternative DNA amplification and cloning reagents that circumvent the use of radioactivity and the need for the single-stranded conformation polymorphism gels. The method is reliable in typing dogs for the class I dog leukocyte antigen (DLA)-88 locus, and through its use, we describe here two new alleles DLA-88*01101 and DLA-88*01201.

Polyamine synthesis and transport inhibition in a human anaplastic thyroid carcinoma cell line in vitro and as xenograft tumors.

Polyamines are essential cellular components for neoplastic transformation and cell proliferation. Antineoplastic efforts that inhibit polyamine synthesis are insufficient to induce cytotoxicity, due to compensatory induction of polyamine transport. Treatment of an anaplastic human thyroid carcinoma cell line (DRO90-1) with a novel polymeric spermine conjugate (polyspermine; PSpm) caused in vitro cytotoxicity and inhibited the growth of xenograft tumors at low concentrations. Similar in vitro antineoplastic effects were noted with two other human anaplastic thyroid carcinoma cell lines. This coincided with inhibition of polyamine uptake and synthetic enzyme activities, with reduced ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAM-DC) but increased spermidine/spermine N1-acetyltransferase (SSAT) activities, as measured in DRO90-1 cells. In subsequent studies using these cells, PSpm was effective in reducing the intracellular levels of all polyamines in vitro, resulting in cytotoxicity that was not reversed by administration of extracellular polyamines. Low-dose PSpm inhibited tumor growth in vivo, but high doses of PSpm potentiated xenograft tumor growth. PSpm degradation products produced with in vivo treatment may be produced that function as substrates for polyamine biosynthesis. These studies suggest that polyamine metabolism inhibition is a viable target for antineoplastic therapy of anaplastic thyroid carcinoma, although the in vivo response to PSpm suggests that this agent will have limited clinical utility.

Restoration of iodide uptake in dedifferentiated thyroid carcinoma: relationship to human Na+/I-symporter gene methylation status.

Disseminated dedifferentiated thyroid epithelial carcinoma, which cannot sufficiently concentrate therapeutic radioiodide, is a terminal disease without any effective systemic treatment or chemotherapy. This is a likely consequence of loss of human sodium-iodide symporter (hNIS) function. We hypothesized that hNIS transcriptional failure in thyroid carcinoma could be consequent to methylation of DNA in critical regulatory regions and could be reversed with chemical demethylation treatment. Analysis of hNIS messenger ribonucleic acid (mRNA) expression in 23 tumor samples revealed that although loss of this expression corresponded to loss of clinical radioiodide uptake, some thyroid carcinomas with hNIS mRNA expression did not concentrate iodide, suggesting additional posttranscriptional mechanisms for loss of hNIS function. In addition, analysis of DNA methylation in CpG-rich regions of the hNIS promoter extending to the first intron failed to define specific methylation patterns associated with transcriptional failure in human thyroid tumor samples. In seven human thyroid carcinoma cell lines lacking hNIS mRNA, treatment with 5-azacytidine or sodium butyrate was able to restore hNIS mRNA expression in four cell lines and iodide transport in two cell lines. Investigation of methylation patterns in these cell lines revealed that successful restoration of hNIS transcription was associated with demethylation of hNIS DNA in the untranslated region within the first exon. This was also associated with restoration of expression of thyroid transcription factor-1. These results suggest a role for DNA methylation in loss of hNIS expression in thyroid carcinomas as well as a potential application for chemical demethylation therapy in restoring responsiveness to therapeutic radioiodide.

Cloning of the human sodium-iodide symporter promoter and characterization in a differentiated human thyroid cell line, KAT-50.

Elucidation of the regulation of human sodium-iodide symporter (hNIS) gene expression is critical to understanding its effects on iodide concentration abilities of thyroid and thyroid carcinomas. To explore this issue, a 1.2-kb portion of the 5'-flanking region of the hNIS gene was isolated and characterized. Transient transfections with chimeric luciferase-reporter constructs into a differentiated human thyroid cell line, KAT-50, as well as non-thyroidal cells, defined an active promoter with tissue-specificity. Reverse-transcriptase polymerase chain reaction analysis for hNIS mRNA expression in normal human tissues was positive in thyroid, salivary gland, omentum, and gallbladder. KAT-50 cells expressed hNIS mRNA and were capable of thyrotropin-responsive iodide uptake in vitro. Despite the failure to exhibit iodide concentration in clinical anaplastic carcinoma tumors, 4 of 5 cell lines from this cancer phenotype expressed hNIS mRNA. Definition of the active promoter provides further insights and tools to uncover new approaches to use of radioiodine for therapy of thyroid carcinomas.

A frequently amplified region in Leishmania contains a gene conserved in prokaryotes and eukaryotes.

A 27.5-kb sequence that is present in an approx. 2-Mb chromosome in Leishmania also occurs as an inverted dimer in a multicopy, 55-kb circular molecule (LD1) in Leishmania infantum ITMAP263. Sequence analysis of a 7100-bp cloned segment from the circular molecule revealed three open reading frames (ORFs). The ORFs are likely to have protein coding function by a number of criteria, including Northern blot analyses. The amino acid (aa) sequences deduced from two ORFs showed no similarity to other sequences in the databases. The C-terminal aa sequence from the third ORF is related (22-29% identity, 57-71% similarity) to a family of genes conserved in bacteria and humans. One member (sfhB) of the gene family in Escherichia coli appears to have a role in regulation of cell growth.